RA is a chronic inflammatory autoimmune disease of unknown etiology. Although several therapeutic modalities exist for the treatment of RA, at present there is no cure. RA affects about 1% of the population worldwide, and 2% of the adults aged 65 and older. RA can start at any age and its prevalence is nearly three times more common in women than men. RA is a disease which shows poor long-term prognosis. Statistics indicate that 80 percent of affected patients are disabled after 20 years, and life expectancy is reduced by an average of 3 to 18 years.
RA is a major cause of disability, morbidity and mortality. RA is characterized by symmetric inflammation of synovial joints that leads to progressive erosions of cartilage and bone. This process attacks the cartilage by following a so-called pannus and erodes the underlying bone by recruiting osteoclasts. Tendons and other body parts are further affected. Since damaged joints are normally irreparable, this often led to permanent incapacitation. For most untreated RA patients, irreversible joint damage occurs within 2 years.
In terms of personal, social and economic costs, the effects of RA are substantial. Recent studies indicate that the mean annual medical cost for each patient was $6300 USD in the United States, $6800 CND in Canada, 4700 in Germany and £2700 in the United Kingdom. The total annual costs for RA care are currently about $2 billion in Canada and $18 billion in the United States; a number that is expected to increase dramatically with an aging population.
Novel ideas for the development of drugs that specifically treat RA with minimal adverse events and costs are urgently needed. The classical paradigm for RA pathogenesis holds that CD4+ T cells mediate joint damage both directly and by driving non-T effector cells to release inflammatory cytokines. Disease modifying antirheumatic drugs (DMARDs) are the mainstay of treatment of RA, however they suffer from limited efficacy, toxicity problems or both. Biological based interventional therapies, such as TNFα antagonists have shown marked anti-inflammatory affects, however there are severe risks associated with their use. Only about 60-70% of RA patients so treated show an ACR20 response (defined as the minimal acceptable response to be achieved for a RA patient to have responded to a drug). The costs of biologicals are about 2 to 7 times that of the DMARDs, however none of these therapies are effective at blocking disease progression.
Gold drugs, as the “gold standard” of therapy, have been used to treat RA for over 78 years to retard and sometimes cause remissions of the disease. The mechanism of gold for antiarthritic activity was based on the active site directed inactivation of the T cell and cysteine (Cys) proteases, particularly cathepsins, by formation of chelate complexes between gold and Cys thiol groups of the affected antigenic peptide and cathepsins. Highly expressed cathepsins as direct contributors to inflammation and cartilage and bone degradation were detected both in synovial membrane and fluid in RA. The activities of cathepsins were significantly correlated with the concentration of inflammatory cytokines such as interleukin-1β (IL-1β) and TNFα. Early clinical data with cathepsin inhibitors showed potential that it may be effective for RA care. The selective inhibition of cathepsins by modification of the thiol moiety may potentially lead to the development of a novel chemotherapeutic treatment for RA. The progress in cathepsin inhibitors design and synthesis has been advanced by the substantial interest of pharmaceutical companies such as Apotex, AstraZeneca, Aventis, Bayer, Bochringes Ingelheim, Celera Genomics, GlaxoSmithKline, Merck, and Novartis.
The new paradigm for RA pathogenesis focuses on an interaction between B cells and CD4+ T cells. Anti-CD20 rituximab based B-lymphocyte depletion (BLyD) therapy has therapeutic potential for patients with RA. BLyD led B cells disappear within days but clinical improvement and autoantibody decline may progress over as long as 9 months. Sustained clinical responses with an impressive improvement was seen for up to 1 year according to ACR50-70 criteria (defined as the good and excellent response to be achieved for a RA patient to have responded to a drug), reported in case reports, open-label pilot studies, and a randomized, double-blind, placebo-controlled trial. BLyD used in combination with DMARDs methotrexate or cyclophosphamide appears to be a reasonable treatment option for refractory RA. These positive responses were associated with a major decline in C Reactive Protein (CRP) and autoantibody levels including all classes of rheumatoid factor (RF) and second anti-cyclic citrullinated peptides (anti-CCP 2). Memory B cells from RA patients were more sensitive to Rituximab than memory cells from normal controls. Defective B cell tolerance checkpoints were seen from patients with RA that may favor the development of autoimmunity. It is likely that the clinical pathology of RA is antibody-mediated and BLyD prevents replenishment from B cells. Qualitative or quantitative differences in B cell commitment in RA pathobiology might have a function in the different responses observed. The effects of BLyD lend increasing support to the idea that both inflammatory effector mechanism and underlying immunoregulatory disturbance in RA are driven by autoantibody rather than by T cells. This realization represents a significant leap forward in understanding the pathogenic mechanism of RA. The generation of rare pathogenic B cell subsets may be a rate-limiting step in the pathogenesis of RA.
Although BLyD shows great promise and reveals mechanism of the disease, there is not yet a long-term strategy for routine use. Chronically maintained depletion with frequently repeated BlyD is unlikely to be a viable option, even in a case with sustained benefit, since such treatment may induce hypogammaglobulinaemia and serious infections. Identifying the relevant antigen that drives the pathogenic mechanism in RA is crucial to form an antigen-based immunointervention, e.g., tolerizing vaccine. It would be preferable to induce the death of autoimmune B cells by antigens with the absolute certainty that said antigens administration will never induce or recall a pathogenic response. Small molecules that specifically discourage the survival of autoreactive B cells in the disease should provide an efficient and cost-effective treatment of RA. Gold thiomalate as the blockade of thiols is a perfect drug for RA because it can induce complete and permanent remission but the problem is that it achieves this very rarely, and more often produces toxicity. There is no reason to not think that if we understand how drugs like gold really work it should not be possible to separate efficacy from toxicity. It is reasonable to hope that further development of strategies targeting pathogenic B-cell clones will extend this toward the original aim of truly long-term remission of RA.
A “window of therapeutic opportunity” has been suggested to be present within the first 3 months of the onset of the disease symptom. Timely intervention is crucial in preventing irreversible joint damage and diagnosis and aggressive therapy during the earliest stages of RA could significantly improve its medical and economic outcome.
The 1987 RA classification criteria of the American College of Rheumatology (ACR) were developed using patients with established RA (mean disease duration close to 8 years) having 91% sensitivity and 89% specificity. RA is routinely defined in clinical practice by the presence of least 4 of the 7 criteria, wherein clinical criteria 1 through 4 must be present for over 6 weeks. These ACR criteria are often not manifested in early RA. RF is the only serological marker included in the ACR criteria. RF can be detected in 50-80% of RA sera, but is frequently present in healthy individuals (especially elderly), and in patients with infections and other autoimmune diseases. Diagnosis of RA using RF testing remains suboptimal. The ACR criteria have served rheumatologists well for decades for established RA but they remain inadequate due to limited usefulness in early RA.
New criteria are urgently needed to shift from classifying established RA to classifying early RA that is projected to become persistent and erosive disease. The new criteria should include reliable serological marker that detects RA in early stages both highly specifically and sensitively. The new measure will lead to a definitive diagnosis of early RA with enhanced ability to prognosis the disease outcome.
The recent discovery of citrullinated protein/peptide (CP) bound autoantibodies as highly specific diagnostic markers for RA has revolutionized the early serodiagnostic evaluation of patients with RA and represents the first commercially available assay that approaches this goal. Posttranslational modification of self-protein with citrullination involvement has been suggested to be a crucial step in the generation of neoepitopes responding to production of RA-specific antibodies. Endogenous peptidylarginine deiminase (PAD) enzymes catalyze the conversion of peptidyl arginine to peptidyl citrulline. Non-coded amino acid citrulline (Cit) has been suggested as the neoepitope of acquired antigenicity of protein through deimination.
Along with the immunological findings on citrullination in proteins, a genetic connection between PADI4 and RA has been reported to be associated in the Japanese and Korean populations. But, the association was not replicated in the Caucasians, Spanish and UK populations. A family based study also showed no association between PADI4 and RA in a white French population. No evidence was found for the association of the PADI4 gene with severity as assessed by erosive outcome or with presence of antibodies against citrullinated antigen in patients with inflammatory polyarthritis.
Antibodies to CP are locally produced in the inflamed synovium, PADI2 and PADI4 are also localized in synovial tissue. These findings suggest a possibility that local citrullination of intra- and extra-articular proteins might be the initial event leading to autoantibody production in RA. CP derived from α-erolase, fibrin, fibrinogen, fibronectin, and vimentin in synovial tissue or joints, keratin, perinuclear factor, and filaggrins in epithelial tissues, type I and II collagen (CI, CII) in cartilage-specific extracellular matrix, and viral peptide in EBV infected B lymphocytes and epithelial cells of the oropharynx, have been suggested as the autoantigen candidates. Besides these, citrullinated asporin, cathepsin D, β-actin, CapZα-1, albumin, eukaryotic translation initiation factor 4, histamine receptor, protein disulfide-isomerase (PDI) ER60 precursor, mitochondrial aldehyde dehydrogenase (ALDH2), and Sp alpha (CD5 antigen-like protein) receptor were identified as the candidate citrullinated autoantigens in RA. Proteomic approach identified 51 (5.2%) of the visualized 990 synovial proteins from patients with RA were citrullinated. Ninety-four (9.5%) of the 990 protein spots were reactive to the RA sera. Interestingly, 30 (31.9%) of the 94 RA sera-reactive spots were CP and 30 (58.8%) of the 51 CP were RA sera-reactive. But, researchers have thus far failed to demonstrate that CP act as driving antigens for the induction of arthritogenicity and autoimmunity in experimental mouse arthritis models. Increased immunogenicity and arthritogenicity were seen when mice were immunized with CP in the presence of adjuvant, and none of the mice investigated developed arthritis. In collagen-induced arthritis however, linear citrullinated peptide (LCP) tolerized mice demonstrated significantly reduced disease severity and incidence compared with controls. Citrullinated fibrin was observed from any synovitis in synovial tissue from RA patients and control patients. There were citrullination of histones and nucleophosmin/B23 in HL-60 granulocytes, and citrullination of keratins and antithrombin in epidermis and plasma. Citrullination was detected in lung specimens of patients with RA-associated IP and patients with IP and control patients. Abnormally accumulated CP was detected in hippocampal extracts from patients with Alzheimers disease. Citrullination of myelin basic protein was observed during development of experimental autoimmune encephalomyelitis. Brain proteins with citrullination were correlated to the pathophysiology of multiple sclerosis brains. CP was also found in Browmans capsules and in obstructive nephropathy. Thus, citrullination is a general phenomenon of posttranslational modification of self-protein. Citrullination is crucial but not essential in induce an autoimmune response with production of RA-specific antibodies. Citrulination is incapable of induce an autoimmune response with production of antibodies unrelated to RA. The profile of citrullination in immunogenicity of the autoantigen has been poorly understood. The origin of Cit-containing autoantigen involved in the induction of RA-specific antibodies remains elucidative.
RA-specific antibodies have been classified into anti-CP (antibody against CP derived from α-enolase, CI, CII, fibrin, filaggrin, keratin, perinuclear factor, or vimentin), anti-LCP (antibody against LCP derived from profilaggrin, filaggrin, CI, CII, or EBV nuclear antigen), anti-CCP1 (antibody against single CCP derived from filaggrin) and anti-CCP2 (antibody against artificial CCPs derived from peptide libraries with non homologous from known proteins) based on capture antigen used to detect them. Anti-CP test showed high specificity (˜100%) at low sensitivity (46%) (n=8941) (Palosuo et al., 1998; Hayem et al., 1999; Vincent et al., 1999; Forslin et al., 2000; Goldbach-Mansky et al., 2000; Menard et al., 2000; Nogueira et al., 2001; Bas et al., 2002; Vincent et al., 2002; Meyer et al., 2003; Saraux et al., 2003; Suzuki et al., 2003; Suzuki et al., 2003; Vencovsky et al., 2003; Dubucquoi et al., 2004; Grootenboer-Mignot et al., 2004; Vittecoq et al., 2004; Auger et al., 2005; Boire et al., 2005; Greiner et al., 2005; Kinloch et al., 2005; Nielen et al., 2005; Suzuki et al., 2005; Chen et al., 2006; Dejaco et al., 2006; Hill et al., 2006; Lopez-Longo et al., 2006; Matsuo et al., 2006; Rodriguez-Mahou et al., 2006; Tian et al., 2006; Vander Cruyssen et al., 2006; Yoshida et al., 2006; Agrawal et al., 2007; Coenen et al., 2007). A similar specificity (˜100%) and sensitivity (48%) were detected when either naturally derived or chemically synthesized LCP were used as capture antigen (n=3668) (Schellekens et al., 1998; Girbal-Neuhauser et al., 1999; Schellekens et al., 2000; Union et al., 2002; De Rycke et al., 2004; Dubucquoi et al., 2004; Hoffman et al., 2004; Low et al., 2004; Burhardt et al., 2005; Koivula et al., 2005; Merlini et al., 2005; Anzilotti et al., 2006; Koivula et al., 2006; Pratesi et al., 2006; Vander Cruyssen et al., 2006; Vander Cruyssen et al., 2007). Using CCP considerably increased sensitivity in comparison with its LCP counterpart without sacrificing specificity, anti-CCP1 were 53% sensitivity at ˜100% specificity (n=2948) (Goldbach-Mansky et al., 2000; Kroot et al., 2000; Schellekens et al., 2000; Bizzaro et al., 2001; Bas et al., 2002; Jansen et al., 2002; Vincent et al., 2002; Bas et al., 2003; Jansen et al., 2003; Meyer et al., 2003; Saroux et al., 2003; Vencovsky et al., 2003; Zeng et al., 2003; Feng et al., 2004; van Gaalen et al., 2005; Vander Cruyssen et al., 2006), and anti-CCP2 were 66% sensitivity at ˜100% specificity (n=19385) (Lee et al., 2003; Pinheiro et al., 2003; Suzuki et al., 2003; Alessandri et al., 2004; Berglin et al., 2004; Bobbio-Pallavicini et al., 2004; Bombardien et al., 2004; Bongi et al., 2004; Correa et al., 2004; De Rycke et al., 2004; Dubucquoi et al., 2004; Forslind et al., 2004; Girelli et al., 2004; Grootenboer-Mignot et al., 2004; Hitchon et al., 2004; Kasapcopur et al., 2004; Kastbom et al., 2004; Lopez-Hoyos et al., 2004; Low et al., 2004; Mikuls et al., 2004; Soderlin et al., 2004; Solanki et al., 2004; Vallbracht et al., 2004; van Gaalen et al., 2004; Vittecoq et al., 2004; Aotsuka et al., 2005; Boire et al., 2005; Burkhardt et al., 2005; Caramaschi et al., 2005; Choi et al., 2005; Dubrous et al., 2005; Femandez-Suarez et al., 2005; Gao et al., 2005; Garcia-Berrocal et al., 2005; Greiner et al., 2005; Hiura et al., 2005; Huizing a et al., 2005; Irigoyen et al., 2005; Kamali et al., 2005; Koivula et al., 2005; Kwok et al., 2005; Limaye et al., 2005; Lindqvist et al., 2005; Mu et al., 2005; Nakamura et al., 2005; Nell et al., 2005; Nielen et al., 2005; Quinn et al., 2005; Raza et al., 2005; Ronnelid et al., 2005; Samanci et al., 2005; Sauerland et al., 2005; Shovman et al., 2005; Sihvonen et al., 2005; Spadaro et al., 2005; Tampoia et al., 2005; Tobon et al., 2005; van der Helm-van Mil et al., 2005; van Gaalen et al., 2005; Verpoort et al., 2005; Alenius et al., 2006; Ates et al., 2006; Atzeni et al., 2006; Benucci et al., 2006; Berglin et al., 2006; Braun-Moscovici et al., 2006; Caspi et al., 2006; Ceccato et al., 2006; Chen et al., 2006; Dejaco et al., 2006; del Val del Amo et al., 2006; Hill et al., 2006; Inanc et al., 2006; Johansson et al., 2006; Koivula et al., 2006; Korkmaz et al., 2006; Linn-Rasker et al., 2006; Lopez-Longo et al., 2006; Matsui et al., 2006; Mewar et al., 2006; Meyer et al., 2006; Mikuls et al., 2006; Panchagnula et al., 2006; Pedersen et al., 2006; Pierer et al., 2006; Redaitene et al., 2006; Rodriguez-Mahou et al., 2006; Russell et al., 2006; Shankar et al., 2006; Spadaro et al., 2006; Tamai et al., 2006; vander Cruyssen et al., 2006; van der Helm-van Mil et al., 2006; Agrawal et al., 2007; Coenen et al., 2007; Forslind et al., 2007; Inanc et al., 2007; Kaltenhauser et al., 2007; Kudo-Tanaka et al., 2007; Ligeiro et al., 2007; Rantapaa-Dahlqvist et al., 2007; Turesson et al., 2007; van der Helm-van Mil et al., 2007).
RA patients can have anti-CP-positive or anti-CP-negative, anti-LCP-positive or anti-LCP-negative, anti-CCP1-positive or anti-CCP1-negative, and anti-McHale CCP2-positive or anti-CCP2-negative phenotypes. In RA patients with at least one of anti-citrullinated antigen, 78% have anti-CP and anti-CCP2 antibodies, 30% have anti-LCP and anti-CCP2, 73% have anti-CCP1 and anti-CCP2; and 12-32% have only anti-CP, 3% have only anti-LCP, 4-19% have just anti-CCP1, and 21-29% have just anti-CCP2. Patients with RA contain at least one of three anti-CP i.e. AFA, AKA and APA, 59% have concordant for all three antibodies, 25% overlap two antibodies, and 18% have only one antibody. About 5% of the statues of anti-CCP2 were changed during 3 years antirheumatic treatment in patients with RA: 2% from negative to positive and 3% from positive to negative. The frequencies of antibodies to citrullinated CapZalpha-1 were 53.3% in RA group, and 36.7% in the RA group where the antigen was non-citrullinated. Antibodies to citrullinated CII were detected in 78.5% of serum samples from 130 RA patients. Antibodies to native non-citrullinated CII were detected in 14.6% of serum samples, all of which were positive for anti-citrullinated CII. The IgG subclass profiles of antibodies against citrullinated fibrinogen in patients with RA are 61% for IgG1, 34.8% for IgG1+IgG2, IgG1+IgG3, or IgG1+IgG4, and 4.2% for IgG1+IgG2+IgG3, IgG1+IgG2+IgG4, or IgG1+IgG3+IgG4. Despite their more specific association with RA, antibodies against citrullinated antigen do have a remarkable variability in the reactive pattern towards different Cit-containing antigen. Anti-citrullinated antigen antibodies are not equivalent as diagnostic markers in patients with RA.
Detection of antibodies against citrullinated antigen has led to a definitive diagnosis of RA up to 14 years before onset of the first disease symptom. The antibodies appear to be highly predictive of the future development of RA in both healthy subjects and patients with undifferentiated arthritis. The activity of antibodies against citrullinated antigen is associated with upregulation of proinflammatory cytokines. The phenotype of RA patients with or without these antibodies is similar with respect to serological parameters of disease activity (CRP, ESR, and WBC) and clinical presentation, but differs with respect to disease course. Patients with at least one of these antibodies have a high predictive value for the development of persistent RA, worse clinical disease, more swollen joints, great radiological progression and joint destruction, severe bone lesions, mortality risk, and demand more effective antirheumatic treatment. Anti-CCP2 test identified more patients with joint damage progression than anti-LCP and anti-CCP1 test. Anti-CP but not anti-CCP2 had best predicted severity in patients with recent-onset or early polyarthritis. Anti-citrullinated antigen antibodies are not equivalent as prognostic markers in patients with RA.
A number of studies have examined the effects of active RA treatments on the serum levels of antibodies against citrullinated antigen. The doses of the antibodies was found as a stable phenotype that remained essentially unchanged after diagnosis and initiation with DMARDs, biological or biological combined DMARDs therapy, as well as clinical and remission progression. RF, but not the antibodies, were associated with clinical treatment efficacy indicating there are two independent autoantibody systems in RA. These results are in contrast to those that found active therapy by biological TNFα blockers including infliximab, adalimumab and etanercept, or by these blockers combined with DMARDs methotrexate resulted in clinical improvements to reflect significant declines in levels of anti-CCP2. BLyD therapies brought clinical responses in responding patients and were correlated with a great decrease in the levels of anti-CCP2. Nonresponding patients did not present any significant variation in anti-CCP2 levels. Antibodies to citrullinated antigen are thought to play an important role in RA pathogenesis.
The DRB1 SE alleles have been suggested to be associated with antibodies against CCP2 and predict severity in RA. Citrullinated peptide featured a high-affinity peptide interaction with HLA-DRB1 alleles. Anti-CCP2-positive RA was exclusively associated with HLA-DRB 1, and anti-CCP2-negative RA was exclusively associated with HLA-DR3. High titres of anti-CCP antibodies were significantly associated with the presence of HLA-DRB1 04/10. Data from a large number of fibrinogen peptides in both native and citrullinated forms, however, found that citrullination was not a prerequisite for binding of peptide to HLA-DR-associated alleles, and citrullinated fibrinogen peptides did not stimulate T cell proliferation more efficiently than their native forms. HLA-DRB1 alleles are most likely associated with anti-CP production because these alleles promiscuously bind fibrinogen peptides. Fifty-five percent of the sera from SE-negative RA patients were positive for anti-citrullinated fibrinogen. Expression of HLA-DRB 1 is not mandatory in order for RA patients to develop antibodies against citrullinated fibrinogen.
If citrullination of self protein is a critical step leading into breakdown in immunological tolerance, the neoepitope created on CP must be more convincingly native and decisive than the epitope mimicked by LCP, CCP1 and CCP2. However, not all proteins harboring Arg residues become reactive with anti-CP after efficient in vitro citrullination. Among 71 15-mer citrullinated fibrin-derived peptides, only 19 of them were specifically recognized by anti-CP. None of immunoreactive CP bears all the epitopes targeted by anti-CP, anti-LCP, anti-CCP1, and anti-CCP2. The sensitivity in detection of RA by anti-CP is distinctively lower than the sensitivity detected using anti-LCP, anti-CCP1 and anti-CCP2 (46% versus 48%, 53% and 66%), but all of them are extremely specific (˜100%), indicating the presence of citrullinated moiety in an antigen is crucial but not sufficient. Patients with anti-CCP2-positive bore citrullinated epitopes with features predictive of the development of severe RA, and in contrast, patients with anti-CCP2-negative bore native epitopes with features predictive of less severe RA. AFA was reacted with the uncitrullinated filaggrin as an antigen and its titer was correlated with clinical parameters in patients with RA. These studies have shown that not all CP/LCP/CCP1/CCP2, nor uncitrullinated proteins/peptides equally react with antibodies against citrullinated antigen. RA-specific antibodies are anti-hapten antibodies, and the hapten is consisted of Cit and non-Cit residues on linear rather than conformational basis. Interrelation between Cit and non-Cit residues and surrounding other AA residues may determine the visibility of Cit residue, non-Cit residue or both Cit and non-Cit residues on an antigen, resulting in variable anti-citrullinated antigen-positive or anti-citrullinated antigen-negative phenotypes in patients with RA.
A chemical composition with limited mass (MW usually less than 1000) is defined as a hapten that does not elicit antibody formation when introduced into a host animal. But, when it covalently couples to a high molecular weight carrier, the resultant hapten-carrier conjugate could elicit in the host animal the formation of antibodies that recognizes the hapten. Posttranslational modification could make protein with acquired autoimmunity via adding autoepitope and elicit antibody against that neoepitope which was created. If the autoepitope is a hapten as small as Cit residue (MW 157.20) it is unlikely that it could completely occupy an entire antigen-binding site of the antibody. The bridge group that protein used to connect hapten could become an additional antigenic part other than the hapten to form the neoepitope and bind the antibody. If the neoepitope was contaminated from an incorrect bridge group, that interference could result in underestimation or overestimation of the antibody. Bridge group recognition is a general property of anti-hapten antibodies having a profound effect on sensitivity and specificity in assays using hapten-carrier as antigen. Encoding the linear epitope recognized by RA-specific antibodies will determine whether Cit, non-Cit AA, Cit residue, non-Cit AA residue, bridge group, or some combination thereof contribute to the overall binding requirements of the epitope.
Major challenges in the diagnosis and treatment of RA remain. Key issues include:
1) The lack of immunogenicity of CP to the immune system. In contrast to antibodies against CP, which have an impressive specificity of nearly 100% in RA patients and <1% of the population develops the antibodies, the presence of CP are not specific for RA but rather are a result of inflammation in everyone's life. Immunity against CP does not cause RA in experimental animal models. Does it suggest there is a process with inclusive citrullination involved in the initiation or perpetuation of autoimmunity or merely reflects ongoing inflammation that has yet to be discovered? Does it imply there is a Cit-containing autoantigen that has yet to be discovered?2) The insufficient antigenicity of citrullinated antigens with existing antibodies. The sensitivity of antibodies to citrullinated antigens is detected in 46-66% of patients with RA. Does this imply that a considerable proportion of patients with RA do not produce anti-citrullinated antigen antibodies or are current capture antigens simply unable to detect them because of incomplete antigenicity?3) The insufficient diagnostic accuracy of antibodies against citrullinated antigens. RA-specific antibodies to citrullinated antigens are highly specific but less sensitive. Does this indicate that Cit residue is the epitope's decisive component responsible for detection of antibodies against citrullinated antigens positive RA? If so, what is the epitope's decisive component responsible for the detection of antibodies against citrullinated antigens negative RA?4) Considering a proportion of patients with RA do not harbor anti-citrullinated antigens antibodies, does this indicate that the presence of the antibodies is not obligatory for the development of RA or that the pathogenic mechanisms underlying anti-citrullinated antigen-positive RA and anti-citrullinated antigen-negative RA are different?5) Considering CCP2 is moderately sensitive and detects a different subgroup of antibodies than CCP1, LCP and CP, does this indicate that the presence of bridge group recognition could result in citrullinated antigen being “visible” or “invisible” for existing specific antibodies, and does this imply that at least part of the anti-citrullinated antigen-positive were ill-defined anti-citrullinated antigen-negative, and anti-citrullinated antigen-positive and -negative do not belong to two independent development processes?6) What is the antigen responsible for the generation of immune response against the citrullinated antigens? Mapping RA-specific antibodies-targeted epitope is important and could lead to elucidate the corresponding antigen puzzle.7) The relationships between specific antibodies production and severe outcomes or therapeutic response in the disease.8) How, when, where and why is the responsible break in immune tolerance generated? Does the immunity cause arthritis in animals?9) The safety and efficacy of expensive new biological therapies.10) Is BLyD a way to treat RA? Selectively depleting B cells involved in RA may be beneficial, as this would prevent production of RA-specific antibodies.11) If RA-specific antibodies are involved in the pathogenesis of RA, does this imply that the antigen-specific interventions could prevent chronic arthritis and long-term joint destruction without the side effects associated with today's treatment regiments?12) Thiol-dependent cathepsins are potential targets for RA therapeutic development. What is the thiol moiety responsible for the development or treatment of RA?
More valuable information may come from follow-up studies on the composition of the citrullinated epitope responsible for the specific occurrence of the autoantibody, the new rheumatoid factor in RA sera. If autoantibody-based classification of early RA represents the subtypes of RA that reflect incomplete epitope being harbored in antigenic substrates, analyses of the molecular basis of their antigenicity could ultimately lead to identifying what antigenic elements participated in the unelucidated epitope. If these individuals belong to a critical mutated neoepitope, it could become a platform for generation of an absolutely sensitive and specific RA test. If the epitope detected antibodies can be confirmed to be associated with the development of the disease, tremendous insight to aid diagnostic and therapeutic strategies of RA in clinical practice would be realized.